PROJECT SUMMARY This proposal investigates the epigenetic mechanism of constitutive activation of pathogenic IL6-STAT3 signaling in Fibro-Adipogenic Progenitors (FAPs) from denervated muscles (DEN FAPs). FAPs are essential cellular effectors of skeletal muscle ability to adapt to environmental perturbations. In response to acute injury, FAPs are activated within a coordinate activation of inflammatory and satellite cells (SCs), and contribute to promote muscle repair. By contrast, in response to chronic injury (e.g. muscular dystrophies) dysregulated functional interactions between FAPs, macrophages (MPs) and SCs lead to impaired regeneration and maladaptive repair by fibrosis and fat deposition. We have recently discovered an alternative pattern of FAPs activation in response to muscle denervation, whereby in the absence of concomitant infiltration of MPs and SC activation, a progressive expansion of DEN FAPs with persistent activation of IL6-STAT3 signaling leads to myofiber atrophy and fibrosis. We posit that a flexible usage of cis-regulatory elements of the genome enables FAPs to adopt different functional phenotypes in response to distinct types of skeletal muscle perturbations, through activation of specific patterns of gene expression. In this regard, the differential activation of IL6 transcription observed during FAP?s response to different types of perturbation (i.e. acute injury vs denervation) provides an opportunity to investigate how dynamic interactions between cis-regulatory elements regulate threshold, magnitude and duration of gene transcription in response to distinct stimuli. We will investigate the hypothesis that in DEN FAPs STAT3 promotes long-range interactions between distal enhancers and a highly conserved regulatory element proximal to the IL6 transcription start site (TSS), to activate constitutive high levels of IL6 transcription and secretion, which in turn drives prolonged activation of STAT3, via autocrine engagement of IL6 receptor. As FAPs are composed of discrete sub-populations (subFAPs) in dynamic equilibrium, we hypothesize the constitutive activation of IL6-STAT3 signaling leads to the emergence of a self- autonomous, dominant subpopulation that accounts for the pathogenic activity of DEN FAPs. The specific Aims will test the hypothesis that STAT3-mediated activation of IL6 super-enhancers is a key event for the establishing a pathogenic feedforward IL6-STAT3 loop. In Aim 1 (3D regulation of IL6 locus in DEN FAPs), we will use chromosome conformation capture (3C)- and ChIP-based technologies to identify and characterize IL6 gene-interacting enhancers in DEN FAPs. In Aim 2 (Detection of DEN subFAPs and their and functional characterization), we will exploit single cell RNAseq to detect DEN subFAPs and establish a functional relationship between IL6-STAT3 hyperactivation and their self-autonomous expansion. In Aim 3 Pharmacological targeting of STAT3-activated super-enhancers in DEN FAPs, we will use pharmacological inhibition of STAT3-activated super-enhancers to determine whether interruption of IL6- STAT3 feedforward loop prevents the pathogenic potential of DEN subFAPs.